Nearly 80% of people with diabetes are overweight or obese, suggesting a causal role for excess lipid accumulation in the development of diabetes. Despite the strong association between islet triacylglycerol (TAG) content and 2-cell dysfunction, little is known about TAG synthesis in islets, and whether it has a mechanistic role in 2-cell dysfunction. The long-term objective of this proposal is to determine the mechanisms by which chronic hyperlipidemia / FA exposure impairs glucose stimulated insulin secretion (GSIS). The hypothesis of this proposal is that chronic hyperglycemia and hyperlipidemia increase GPAT1- initiated glycerolipid synthesis in 2-cells and consequently diacylglycerol (DAG) content, which activates PKC5 and impairs GSIS. This proposal outlines the approaches to determine how nutrition regulates GPAT and TAG synthesis in islets and to determine which glycerolipid signal mediates FA-impaired GSIS through the following two aims: SPECIFIC AIM 1: Determine how islet TAG synthesis is regulated and how it plays a role in FA- impaired GSIS. To determine how enzymes of glycerolipid synthesis are regulated in islets, wildtype, Gpat1-/-, and Gpat4-/- mice will be fed a control diet or a high-fat diet to induce hyperglycemia and hyperlipidemia. Changes in Gpat expression and activity will be determined in isolated islets by RT-PCR and an enzymatic activity assay, respectively. Islet glycerolipids, LPA, PA, DAG, and TAG, will be quantified by LC-MS to determine how islet glycerolipid content and composition corresponds to chronic nutrient-enhanced GPAT activity or lack of Gpat-1 or -4, the major GPAT isoforms in islets. GSIS will be measured to assess the effects of manipulating TAG synthesis on 2-cell function. SPECIFIC AIM 2: Determine which lipid intermediates of glycerolipid synthesis mediate FA-impaired GSIS. Enzymes of the TAG synthetic pathway will be systematically inhibited in INS-1 cells to increase or diminish content of LPA, PA, and DAG in order to determine which lipid intermediate impairs GSIS. To determine whether DAG impairs GSIS by activating PKC5, PKC5 activation will be determined by Western blot. PKC5 activation will be inhibited to determine whether impaired GSIS is mediated by PKC5 during chronic nutrient excess. PUBLIC HEALTH RELEVANCE: Nearly 80% of the 23.8 million people in the United States that have diabetes are overweight or obese suggesting a causal role for excess lipid accumulation in the development diabetes. The studies outlined in this proposal will establish a direct mechanism between lipid accumulation in islets and diabetes.